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Takusagawa’s Note Chapter 26 (Summary) Summary of Chapter 26 1. Translation (protein biosynthesis) requires mRNA as template, ribosome, amino acids, tRNA, enzymes, factors, ATPs, and GTPs. 2. tRNA • tRNA molecules contain several modified bases, such as pseudo-uridine (ψ). All four bases are modified. 3. Ribosomes • Ribosomes are composed of two ribonucleoprotein subunits. Each subunit contains rRNAs (~2/3) and proteins (~1/3). • Prokaryotic ribosome 70S = 50S (L subunit) + 30S (S subunit) • Eukaryotic ribosome 80S = 60S (L subunit) + 40S (S subunit) • In general, S-subunit involves in ribosomal recognition processes such as mRNA and tRNA binding, whereas L-subunit involves in the catalytic reaction of polypeptide chain elongation. 4. mRNA • The template (mRNA) is read 5’→3’ direction, and a protein is synthesized N→C direction. • Both 5’- and 3’-ends of mRNA are non-coding region. • Initial codon is AUG, and stop codons are UAA, UAG, and UGA. 5. Translation process 1. Activation of amino acids • A specific amino acid is attached on the 3’-ACC receptor arm by a specific aa-tRNA synthetase. Aminoacyl − tRNA synthetase → Aminoacyl-tRNA + AMD + PPi Amino acid + tRNA + ATP ← • Aminoacyl-tRNA (aa-tRNA) is called charged tRNA, whereas tRNA itself is called uncharged tRNA. 2. Initiation • Both prokaryotic and eukaryotic ribosomes have three tRNA binding sites. 1. P site = Peptidyl-tRNA binding site 2. A site = Aminoacyl-tRNA binding site 3. E site = Exit site of uncharged tRNA Prokaryotic system • The 1st amino acid is always formyl-Met at the initiation codon AUG. • Met- tRNA Met is formylated by N10-formyl THF to fMet- tRNA Met f f • mRNA has a purine-rich region (Shine-Dalgarno sequence) at 10 base upstream from the initiation site. The pyrimidine-rich region of 16S rRNA of ribosome binds to ShineDalgarno sequence. • Initiation process: 3 initiation factors, IF-1, IF-2, IF-3, and fMet- tRNA Met , ribosome, mRNA, f GTP are involved. 1. IF-1 and IF-3 bind to 30S subunit and dissociate it from 50S subunit. 2. 30S subunit complexed with IF-2·GTP and fMet- tRNA Met binds at the initiation codon f and Shine-Dalgarno sequence. This is called 30S initiation complex. 3. Hydrolysis of GTP on IF-2 causes the conformational changes in 30S subunit which result in release of IF-1, IF-2, and IF-3, and in attachment of 50S subunit. This is called 70S initiation complex. fMet- tRNA Met is at P-site. f Eukaryotic system 1 Takusagawa’s Note Chapter 26 (Summary) • The initiation processes are quite similar to those of prokaryotic system except for 1. Additional initiation factor (eIF-4) which binds to 5’ Cap of RNA is required. 2. No Shine-Dalgarno sequence on mRNA. 3. Elongation • The amino group of aa-tRNA in A-site attacks the C-terminal carbonyl carbon of peptidyltRNA in P-site. Thus, the polypeptide is transferred on the aa-tRNA. Prokaryotic system • Three elongation factors (EF-Tu, EF-Ts, EF-G) involved in the processes. The processes are: 1. aa-tRNA binding: EF-Tu forms a ternary complex with GTP and aa-tRNA, and helps aatRNA to bind ribosome by using GTP hydrolysis energy. After aa-tRNA binding to the A site of ribosome, EF-Tu·GDP is released into solution. EF-Ts removes the GDP from the EF-Tu·DGP by binding the GDP binding site of EF-Tu. ET-Ts is replaced with GTP for the next cycle of amino acid attachment. 2. Transpeptidation: Polypeptide on peptidyl-tRNA in P-site is transferred to the tip of amino acid of aa-tRNA in A-site. 3. Polypeptide translocation: EF-G·GTP binds to ribosome and hydrolyzes its GTP to GDP. The hydrolysis energy is used for releasing EF-G from ribosome, transferring uncharged tRNA in P-site to E-site to expel it, transferring peptidyl-tRNA in A site to P-site with mRNA, and shifting a new codon into A site. • Puromycin is an aa-tRNA analog which binds to A-site and receives the polypeptide from peptidyl-tRNA, but cannot translocate it to P site because no tRNA. Eukaryotic system • The processes are quite similar to those of prokaryotic system. Differences are: EF-Tu & EF-Ts are replaced with eEF1, and EF-G is replaced with eEF2. • Eukaryotic elongation factors are functionally equivalent to those of prokaryotic elongation factors, but not exchangeable. 4.Termination • Three releasing factors (RF-1, RF-2, RF-3) involve. 1. When one of stop codons are shifted into A-site, RF-1 or RF-2 binds A-site with RF-3. 2. Binding RFs to A site stimulates hydrolysis of peptidyl-tRNA, and the polypeptide is released into solution. 3. Hydrolysis of GTP on RF-3 leads to release RFs, uncharged tRNA and mRNA from ribosome. 6. Transcription and translation are simultaneously taken place since mRNA is synthesized 5’→3’ direction, and mRNA is read 5’→3’ direction for protein synthesis. 7. Translational accuracy • Translational error rate is very small (~ 10-4 per codon), because there are two check mechanisms. 1. Initial recognition: Codon-anticodon binding energy discriminates between cognate and noncognate tRNA. 2. Proof-reading: After GTP hydrolysis, EF-Ts·GDP and aa-tRNA are separated. The dissociation constant of EF-Ts·GDP from ribosome is independent from nature of aatRNA, but the dissociation constant of aa-tRNA from ribosome depends on nature of aatRNA. Noncognate aa-tRNA has much high dissociation constant than cognate aa-tRNA. Thus, noncognate aa-tRNA dissociates before EF-Tu·GDP does. 8. Protein biosynthesis inhibitors 2 Chapter 26 (Summary) Takusagawa’s Note • Most useful antibiotics inhibit prokaryotic enzyme activities, whereas strong toxins inhibit eukaryotic enzyme activities. • Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor eEF-2 by NAD+. 9. Genetic codes • Normal amino acids are 20, combinations of two nucleotides are only 42 = 16. Thus, the codons are composed of three nucleotides, 43 = 64. • Initially poly(U), poly(A), poly(CU) were used as mRNA, and thus produced poly(Phe), poly(Lys), and poly(Ser-Leu), respectively. • Later 64 codons are determined. All amino acids except for Met and Trp have more than one codons. • Terms: Synonyms = More than two codons specify the same amino acid. Wobble base-pairing = Modified bases in the anti-codon can form not only WatsonCrick base-pair, but also non-Watson-Crick base-pair (Wobble base-pair). Wobble = A tRNA binds more than two different codons by wobble base-pairing. • General rules between codon and amino acid assignment 1. XXY and XXZ specify the same amino acid. 2. XYC and XYU specify the same amino acid. 3. XYA and XYG specify almost the same amino acid. 4. YXX and ZXX specify amino acids with similar character. 5. The modified bases in tRNA make always a base-pair with 3rd base of codons. 10. Nonsense and missense suppressions • If a mutation changes a normal codon to nonsense codon (stop codon), protein synthesis is stop at the mutation site. This problem is rescued by a second mutation on another part of gene (tRNA gene). The mutated tRNA carrying the specific amino acid and continue the protein synthesis. This process is called nonsense suppression. • If a base is inserted between codons, the protein synthesis is completely mixed up after that insertion. This problem is rescued by mutating the tRNA gene to produce a tRNA which recognizes four bases. This process is called missense suppression. 11. Control of translation • Prokaryotic mRNAs have relatively short lifetime (a few minutes). Thus no controls system is necessary for translation. • Eukaryotic mRNAs have relatively long lifetime (hours to days). Thus, a proper control system is needed to regulate the translation activity. • One control site is: eIF-2·GDP + GTP ↔ eIF-2·GTP + GDP catalyzed by eIF-2B. • Example, globin synthesis: • Hemoglobin is composed of hemes and globin proteins. • Globin synthesis is regulated by heme availability, i.e., high [heme] activates the synthesis and low [heme] inhibit the synthesis. • At low [heme], inactive pro-HCR (heme-controlled repressor) is converted to active HCR. • HCR catalyzes phosphorylation on eIF-2. • The phosphorylated eIF-2 makes a tight complex with eIF-2B. Thus, [eIF-2B] is reduced. Thus, the GDP↔GTP exchaine rate on eIF-2 is slow down. 12. Protein degradation • Cells, which lack lysosomes, selectively degrade abnormal proteins by cytosolically based ATP-dependent proteolytic system (Proteosomes). • Ubiquitin and ubiquitin-conjugating enzyme selects condemned protein and send it to proteosome to degradation. 3